Correction device for an optical arrangement and confocal microscope with such a device

ABSTRACT

A correction device for an imaging optical arrangement exhibiting a light path ( 1 ), in particular for a microscope, that exhibits at least one plane-parallel transparent plate ( 9 ), which is held in a mounting plate in the image beam path ( 1 ) and is propelable around at least one axle in a tipping and/or a swiveling motion, in order in adjust a definite parallel misalignment of the beams in the image beam path ( 1 ) by a change in the tipping situation of the plate ( 9 ). A confocal microscope with such a correction device exhibits a confocal screen ( 4 ), which illustrates a specimen mark ( 10 ), whereby the plane-parallel plate ( 9 ) is placed in front of the detector unit ( 2 ) in the light path ( 1 ), in order to center the illustration of the aperture diaphragm on the detector unit.

The invention refers to a correction device for an optical arrangementthat exhibits a light path, for example for a microscope. It speciallyrefers to a confocal microscope with such a correction device.

Optical arrangements, like for example microscopes, confocal microscopesor laser scanning microscopes regularly exhibit adjustable elements, inorder to adjust different operating conditions. Thus it is well knownfor example, to exchange filters or color dividers, in order to be ableto work with different lighting wavelengths or to evaluate differentfluorescence radiation in different wavelength ranges. The adjustment orchange mechanisms must be implemented regularly, mechanically and veryexpensively and with high precision, in order to keep the chances ofunwanted disturbances of the light path during element change or elementadjustment as small as possible.

This problem is faced especially in confocal microscopes or laserscanning microscopes, in which a confocal screen with a detector unit isused, that either contains a detector screen on its side or acts as one.Since even with very large mechanical expenditure for the changeable oradjustable elements, influences of the light path for example by tippingerrors or wedge errors at the optical element can never be completelyruled out, partially cost-effective correction mechanisms are providedin the state of technique, to change the light path of the image withsuch pinhole-objectives.

Thus in DE 101 47 481 A1 for example an adjustable confocal screen for alaser scanning microscope is described, that makes a displacement of theaperture possible, to be able to shift the confocal screen appropriatelyin tipping errors or wedge errors that are caused by adjustment orchange of optical elements, so that an optimal image is always formed inthe confocal microscope. The DE 101 07 210 C1 describes a similarapproach, which likewise adjusts relevant elements in the opticalarrangement. There, in a confocal microscope a focusing lens in thearrangement can be shifted transverse to the Z-axis of the light path.It can also be used to bring about an adjustment of the image in theconfocal microscope.

It is thus common in the approaches of the state of the techniques, tochange the optical arrangements in the confocal microscope, i.e. in theoptical imaging arrangement—either by the change of the location of aconfocal screen with respect to the object to be imaged or by theadjustment of other imaging elements of the imaging optics. Apart from arelatively large mechanical/optical effort necessary in theseapproaches, there exists a fundamental problem in this principle pursuedin the state of the techniques, that the reproduction ratios are nolonger comparable from time to time. A laborious new calibration ofimaging scales can become necessary.

Therefore the purpose of this invention is to provide a correctiondevice for an optical imaging arrangement, with which a correction canbe applied without adjusting the optical image itself, especiallywithout having to adjust the optical elements.

According to the invention this task is solved with a correction devicefor an imaging optical arrangement exhibiting an optical path, whereinthe device exhibits at least one plane parallel transparent plate, thatis held in a mounting plate in the optical path and is propelable aroundat least one axle by means of the mounting plate in a tipping movementand/or swiveling, in order to adjust a definite parallel misalignment ofthe light path by change of the tipping situation of the plate.

The invention especially provides for a confocal microscope, whereby themicroscope focuses a selected specimen area on a confocal screen, towhich a detector unit is subordinate and whereby the plate is placedfirst in the optical path of the detector unit, in order to center theillustration.

According to the invention the correction device has the advantage thata simple compensation or correction of errors developing in the image ofthe optical arrangement is possible. Especially simple environment orsystem temperature, radiation used by changeable or mobile elements inthe arrangement, color defects due to wavelength or wavelength rangescan be corrected. Thereby depending on requirement a tipping and/or aswiveling plate with one axle can be sufficient. If one would like toplan a two axle parallel misalignment, one can either use two axletipping and/or swiveling plates, or one can plan a two platearrangement, one axle tipping and one swiveling. It is essential to theinvention that the plane parallel plate can be tipped with the mountingplate in a defined and known way in the light path. For a two-axleadjustment each combination of tipping and swiveling is suitable. Acombination of a tipping and swiveling movement is mechanically andrelatively simple to realize and has surprisingly no disadvantagesdespite the shifting of the plane parallel plate along the Z-axis thatarises during the swiveling.

The correction brought about by the device can be done by a usermanually, e.g. with an adjustment in the works. However, furthertraining with a servo unit is particularly preferable, which records atleast one operating parameter of the optical arrangement and whichadjusts the tipping situation depending on the value of the operatingparameter. The tipping situation can be put into calibration tables, forexample. Also it is possible to optimize a correction by adjusting thetipping situation via active automatic control loops permanently andregularly or on requirement. For such an arrangement it is preferentialto plan an automatic control loop that uses the tipping situation of theplate as correcting variable, in order to balance the described effectson the imaging optical arrangement. So, a possibly existing temperatureor long-term drift error can be balanced in a simple manner in theoptical arrangement.

Since it is well known that the parallel misalignment by a planeparallel plate depends on the refractive index of the transparent diskmaterial, color transverse errors can develop by a wavelength dependentparallel misalignment due to a dispersion of the disk material inpolychromatic radiation in the light path of the optical arrangement. Bystructuring the plane parallel plate from one or several sub panels onecan compensate such color transverse errors caused by the plane-parallelplate.

According to the invention the correction device can be also adjustedfor the correction of varying color transverse errors of the opticalimage that are dependent on the operating conditions. For example, if anoptical arrangement is able to work with different wavelengths then awavelength-dependent and thus operating condition-dependent colortransverse error can occur. According to invention the correction devicecan then adjust the plane parallel plate depending on the wavelengthrange used in the optical arrangement and the resulting color transverseerrors, so that in the final result despite operation with differentwavelength ranges an unchanged optical image is focused in thearrangement. Naturally for this correction again, as previouslymentioned, a suitable servo unit can be used, which can also exhibit anautomatic control loop.

The requirements of the accuracy or sensitivity, with which the drivehandles the mounting plate, can also be preset, like the acceptableparallel misalignment range via the thickness of the plane parallelplate.

The correction device according to the invention reduces, as previouslymentioned, the requirements of adjustable optical elements in thefocusing optical arrangement. This advantage is particularly importantin the case of the already mentioned confocal microscope. In a confocalmicroscope a selected specimen area (Spot) is usually lit up and focusedon a confocal screen in form of a so-called pinhole objective, followedby a detector. The radiation transmitted by this screen arrives with orwithout intermediate image on a detector; the detector can also serve asa confocal screen. The illumination can happen in a linear orpunctiformly pattern.

Care must be taken to focus the specimen area completely on the confocalscreen plane in the pinhole objective. This is above all made moredifficult by the fact that a confocal microscope exhibits regularlyexchangeable beam splitters, with which an adjustment of the microscopefor different applications takes place, i.e. a change of the irradiatedor selected wavelengths. The optical elements capable of being activatedindividually are accompanied by tipping or wedge errors, which can bereduced only with large effort in such a way that they do not disturbthe image in the microscope. The same applies to changes of temperatureor of long-term drift. The correction device according to the inventionpermits the realization of a confocal microscope, with which errorscaused by changing optical elements can be simply corrected withoutinterfering with the optical image. Additionally, the correction devicecan also be adjusted between the confocal screen and detector and so theoptical path between screen and detector can be corrected suitably.

Although in the case of confocal microscopes that use a pinholeobjective before a locally non-resolving detector for detection, thecorrection already facilitates the mechanical requirements regarding theoptical elements capable of being activated, the saving of effort isparticularly noticeable, if the confocal microscope covers a locallydisintegrating detector. This is for example the case with line-scanninglaser scanning microscope that uses a slit diaphragm as pinhole screenbefore a detector line. It is then possible, to balance via acorresponding adjustment of the tipping situation of the plane paralleltransparent plate, both a compensation of deviations perpendicular tothe slit diaphragm and also a compensation of deviations parallel to theslit diaphragm.

In the first case it is guaranteed that the light coming from thespecimen meets the slit diaphragm accurately and is not off-center aboveor below the slit diaphragm. In the second case it is guaranteed thatthe light coming from the specimen meets the line detector correctly andthere is no pixel misalignment between pictures of two detectionchannels in the system, each exhibiting its own line detector forexample. Thus the confocal microscope according to the invention canreach a sub pixel accurate image registration during multi-channeltraining.

The problem with a slit diaphragm that deviates perpendicularly to thedirection line is solved in the confocal microscope by the fact that nowa narrow detector line can be used, without necessitating a movement ofthe slit diaphragm and detector. The unnecessary loss of light flux andthe consequent reduction of the signal-to-noise ratio in case of amisalignment (caused by tipping and wedge errors of changeable elements)with a lowering following an increase in resolution of the slitdiaphragm can be avoided.

Since the tipping or wedge errors of optical elements that can beactivated individually are usually reproducible, the tipping situationof the transparent plane-parallel plate can be selected in a simplemanner. With change of an optical element that can be activated, only adefinite drive action of the plane parallel transparent plate isnecessary in order to adjust the tipping situation newly required forthe desired configuration of the microscope. Therefore a furthertraining of the microscope according to the invention is preferential,in which the change or adjustable elements in the light path areprovided and which records the submission and a configuration of changeor adjustable elements as operating parameters and adjusts the tippingsituation to be dependent on the value of the operating parameter.

An example of such a parameter, with which not only the misalignment ofthe optical image in relation to the pinhole screen but also a colortransverse error is balanced, favorably provides for the usage ofradiation of different wavelengths in the optical path of the microscopewhereby the servo unit records the wavelength in the light path as theoperating parameter and adjusts the tipping situation accordingly. Thenfor a confocal spectral multi-channel microscope one or moreplane-parallel plates are first placed before the detector for eachdetection channel and the tipping situation of the plane-parallel plateis adjusted by the servo unit to be also dependent on the wavelengthand/or the wavelength range of the plate directed toward the detector inthe current channel.

One gets to use a confocal microscope comfortably, if an automaticcontrol loop is provided that maximizes the radiation intensity at thedetector unit, and/or minimizes the picture misalignment by adjustingthe tipping situation of the plane-parallel plates as a correctingvariable. Thus long-term effects or temperature changes involvingmisalignments, can be corrected at any time without a servicetechnician.

Therefore an implementation is preferential, with which the aperturediaphragm and the detector unit are trained as confocal split diaphragmand as detector line respectively and whereby the tipping situation ofthe plane-parallel plate is two axle adjusted such that the picture ofthe slit diaphragm is centered two-axle on the detector line.

The invention is further described below with reference to the figure asan example. The figures illustrate:

FIG. 1 a schematic representation of a detector arrangement of a laserscanning microscope,

FIG. 2 a schematic representation clarifying the need for correctionduring the detector arrangement of FIG. 1,

FIG. 3 a schematic representation of a plane parallel plate in thedetector arrangement of FIG. 1 and

FIG. 4 a perspective representation of the plane-parallel plate of FIG.1 with motor drive.

FIG. 1 shows schematically a detector arrangement 1 for a Laser scanningmicroscope. The detector arrangement 1 exhibits a CCD line 2, which ismerged via a color divider 3 into the light path of the (not representedfurther) laser scanning microscope. The color divider 3 is changeable,in order to be able to record radiation of different wavelength rangeswith the detector arrangement 1. The adaptability by the changeablecolor divider 3 can be given regarding the (excitation) radiation usedin the laser scanning microscope and also regarding (fluorescence)radiation.

The CCD line 2 receives radiation via the color divider 3, which fallson the CCD line 2 by a slit diaphragm 4 working as aperture diaphragm.

The slit diaphragm 4 forms a pinhole objective of the detectorarrangement 1 together with an afore-arranged round optics 5 as well asa likewise afore-arranged first cylinder lens 6 as well as a subordinatesecond cylinder lens 7, whereby the pinhole is realized here by thesplit diaphragm 4. Thus the laser scanning microscope is a line scanningmicroscope, in which a confocal or at least partly-confocal imaging of arectangular or linear range (line) of a specimen takes places by meansof the pinhole objective and/or the detector arrangement 1 on the CCDline 2.

The specimen is illuminated for fluorescence excitation, which isconfocally imaged, is schematically represented as specimen mark 10 inFIG. 1. In order to avoid an unwanted detection of excitation radiationreflected in the system at the CCD line 2, another block filter 8 havingsuitable spectral characteristics is connected before the secondcylinder lens 7, in order to let only desired fluorescence radiationarrive at CCD line 2.

A change in the color divider 3 or the block filter 8 brings aboutinevitably a definite tipping or wedge error while turning. The colordivider can inject an error between specimen line 10 and slot 4, blockfilter 8 can inject an error between slit diaphragm 4 and CCD line 2. Inorder to prevent the need for a readjustment of the situation of theslit diaphragm 4 and/or the CCD line 2, a plane-parallel plate 9 isarranged between the round optics 5 and the slit diaphragm 4, i.e. inthe image beam path between the specimen mark 10 and CCD line 2 whichcan be brought into different tipping positions under the control of thecontroller C. The plane-parallel plate 9 is attached in a suitable (notrepresented in FIG. 1) mounting plate, which will be described later inFIG. 4.

The plane parallel plate 9 causes a parallel misalignment, which isdrawn in FIG. 1, of the Z-axis OA. This parallel misalignment can beseen schematically also in FIG. 3, which concerns (described later) animplementation form of a two-part plane-parallel plate 9. The luminousbeams E diagonal to plate 9 breaking in to the disk surface withdraw astransferred luminous beams A. Without plane-parallel plate 9 there wouldbe the falling beam, drawn dashed in FIG. 3.

A change of the tipping position of the plane-parallel plate 9 makes itpossible to adjust the situation of the the specimen line opposite theslit diaphragm 4 (as well as with the usage of the plate 9 after theslit diaphragm alternatively also the situation of the slit diaphragm 9to the CCD line 2 acting as screen) such that for given conditions inthe light path, which may themselves change by changes of the colordivider 3, always an optimal, i.e. two axle centered situation is given.This is illustrated in FIG. 2, which shows the projection of the slitdiaphragm 4 to the specimen line 10 in plan view. As illustrated, due toa tipping or a wedge error, which can be caused by for example by thecolor divider 3 or the block filter 8, a misalignment dx adjusts itselfin x-direction dx and a misalignment dy in the y-direction between slitdiaphragm 4 and specimen line 10.

The consequence of misalignment dx is that the signal-to-noise ratio isunnecessarily worsened. If one would like to improve the dissolution ofdepth in the confocal microscope by lowering the slit diaphragm 4, i.e.by reducing its expansion in x-direction, it can happen that with amisalignment dx, which is larger than the half height of the specimenline 10 no more radiation arrives at the CCD line. The misalignment dxhas then the consequence that the dissolution of depth attainable withthe laser scanning microscope is actually smaller than is actuallyattainable with the equipment. The same applies to the alternative orcumulative variations in the adjustment of split diaphragm 4 and CCDline 2.

The adjustment of the specimen mark 10 in relation to the slit diaphragmis attained by adjusting the tipping situation of the plane parallelplate 9 such that no surface ranges of the CCD line 2 remainunnecessarily unirradiated when seen in x-direction.

On the other hand the misalignment dy causes the fact that the localinformation recorded in y by the CCD line 2 does not correspond to theactual emission or reflection conditions at the specimen mark 10.Artifacts or a misalignment in the image can be the result. Theadjustment of the tipping situation of plate 9 makes it possible tominimize the misalignment dy preferably even bringing it to zero so thatthe split diaphragm 4 is centrically on the CCD line 2 and is pixels ofthe CCD line 2 are correctly illuminated. This is important inparticular if the laser scanning microscope exhibits several detectorarrangements 1, which select different color channels via differentcolor dividers 3. Since due to the individual adjustments of thedetector arrangements 1 with their color dividers 3 differentmisalignments dy would be present, an error would be the result in sucha multi-channel laser scanning microscope in the allocation of theindividual color channels in a compound picture.

Depending upon wavelength or wavelength range evaluated in the detectorarrangement 1, the pinhole objective of the detector arrangement 1 canexhibit a different color transverse error. Same applies to the elementsarranged before detector arrangement 1, for example the color divisor 3or other optics lying on the Z-axis OA. By the adjustment of the tippingsituation of the plate 9 this color transverse error can be compensatedpurposefully. The controller C steers plate 9 in a tipping situation,whereby each one in the wavelength range and/or each wavelength, forwhich the detector arrangement 1 can be used, is assigned with its owntilting situation.

If in the detector arrangement 1 relatively wide-band radiation isguided, the plane parallel plate can cause a color transverse error, ifthe dispersion of the transparent material of the plane-parallel plate 9is such that a wavelength-dependent misalignment of the failing raybundle A is opposite to the incident luminous beam E. For compensationthe structure of the plane parallel plate 9 represented in FIG. 3consists of two sub panels 9 a, 9 b. The materials of these sub panels 9a, 9 b are different and selected in such a way that in the wavelengthrange, for which the detector arrangement 1 is appropriate, dispersioncaused by color transverse errors if possible cancel themselves. Forexample the subpanel 9 a causes for shorter wavelengths a strongermisalignment than the sub panel 9 b; the reverse applies to longerwavelengths. Thus a compensation of the color transverse error of theplane-parallel plate 9 is attained. For the production of ancolor-independent or aimed color-dependent parallel misalignment alsotwo separated tippable plates with diversion moving in oppositedirections and from materials with different dispersion can be used.

The controller C adjusts the tipping situation of the plate 9 to thedefault of a user, after evaluation of the current configuration (inparticular also environment or equipment temperature or other externalmeasured variables) of the Laser scanning microscope or in continuous orintermittently running control procedures. In the case of a regulationthe tilting situation of the plate 9 is used as correcting variable. Asregulated size the radiation intensity or the picture misalignment onthe CCD line 2 can be evaluated in a calibration step.

The drive 11 steered by the controller 9 is represented in FIG. 4. Asillustrated, the plane-parallel plate 9 is adjusted by means of twostepping motors 12, 13 by the x and/or y axis. The adjustment of thex-axis is a tipping motion with an axis of rotation in the center ofplate 9. The turn around the y-axis is a swiveling around an axle lyingoutside of the plate.

For tipping around the x-axis a retaining plate 14 is provided, ontowhich a pair of leaf springs 5 is screwed, which fasten a framework 16,in which the plane-parallel plate 9 is provided. The leaf springs 15specify the tipping axle. They press one roll 17 fastened at theframework 16 on a cam disc 18, which is propelled by the stepping motor12, which likewise sits on the retaining plate 14. Depending on positionof the cam disc 18 the role 17 and the framework 16 are thus steereddifferently, by which the tipping of the plate 9 is attained around thex-axis.

The retaining plate 14 is for its part an arm of a lever 19, which isswiveling around a drag axis 20. The drag axis 20 represents the axlefor the movement of the plate 9 around the y-level. The other arm 21 ofthe lever 19 carries a role of 22 at its end, which rests against a camdisc 23, which is propelled by the stepping motor 13. Just as the leafsprings 15 press the role 17 on the cam disc 18, a spring element isintended at the drag axis 20, which presses the role 22 on the cam disc23.

By control of the stepping motors 12, 13 the controller C, which isconnected via not any further illustrated lines with the steppingmotors, can adjust the tipping and/or swiveling situation of the planeparallel plate 9 in the light path of the detector arrangement 1 usingthe motor. By the incremental control of the stepping motors 12, 13 thecurrent position of the plate 9 at each point of period of operation iswell-known to the controller C in combination with a reference positionstarted at the operating beginning, so that the position of the plate 9can be used in an automatic control loop can be used as correctingvariable and/or can be adjusted in accordance with stored defaults.

1-14. (canceled)
 15. Correction device for an imaging opticalarrangement (1) having a beam path, comprising: at least oneplane-parallel transparent plate (9), a mounting plate (11) in the imagebeam path, wherein the mounting plate (11) holds the plate (9) in theimage beam path and moves the plate (9) in at least one of a tiltingmovement and a swiveling around at least one axis, in order to adjust adefinite parallel misalignment (dx, dy) of the beams in the light pathby change of the tilting situation of the plate (9).
 16. Deviceaccording to claim 15, wherein the imaging optical arrangement (1) is amicroscope.
 17. Device according to claim 15, wherein the plate (9) hasat least one of a tilting and swiveling movement around at least twoaxes.
 18. Device according to claim 15, comprising at least twoplane-parallel transparent plates (9), each of the plates (9) beingtiltable around one axis.
 19. Device according to claim 15, furthercomprising a servo unit (C), which records at least one operatingparameter of the optical arrangement (1) and which adjusts the tiltingsituation to be dependent of the value of the operating parameter. 20.Device according to claim 15, further comprising an automatic controlloop (C) that uses the tilting situation of the plate (9) as acorrecting variable.
 21. Device according to claim 15, comprising twoplane-parallel transparent plates (9), wherein the plates (9) areindependently ovable and are made from materials of differentdispersion, in order to adjust one of a color-independent and aimedcolor-dependent parallel misalignment.
 22. Device according to claim 18,whereby at least one plate (9) comprises two sub panels (9 a, 9 b) andis made with materials of different dispersion, in order to compensatecolor transverse errors in the light path.
 23. Confocal microscopehaving a beam path and comprising: a confocal screen (4), a detectorunit (2) following the confocal screen (4), and a correction deviceaccording to claim 15 for focusing a selected specimen range (10) on theconfocal screen (4), wherein the plate (9) is placed first in the lightpath of the detector unit (2), in order to center the illustration ofthe selected specimen range (10) on one of the detector unit (2) and anillustration of the screen (4) on the detector unit (2).
 24. Microscopeaccording to claim 23, wherein the detector unit covers a locallyresolved detector (2).
 25. Microscope according to claim 23, furthercomprising changeable or adjustable elements (3) in the light path, anda servo unit (C), which records at least one operating parameter of theconfocal microscope and which adjusts the tilting situation to bedependent of the value of the operating parameter, wherein the servounit (C) records a configuration of the changeable or adjustableelements (3) as operating parameters.
 26. Microscope according to claim25, wherein the light path guides radiation of different wavelengths andthe servo unit (C) records the wavelength in the light path as anoperating parameter.
 27. Microscope according to claim 23, furthercomprising further comprising an automatic control loop (C) that usesthe tilting situation of the plate (9) as a correcting variable, whereinthe automatic control loop (C) maximizes the radiation intensity at thedetector unit (2) and/or minimizes an image misalignment.
 28. Microscopeaccording to claim 23, further comprising a servo unit (C), whichrecords at least one operating parameter of the optical arrangement (1)and which adjusts the tilting situation to be dependent of the value ofthe operating parameter, and wherein the confocal screen is designed asa split diaphragm (4) and the detector unit is designed as detector line(2) and wherein the tilting situation is two-axle adjusted such that theimage of a specimen line is centered on the slit diaphragm (4). 29.Microscope according to claim 23, further comprising a servo unit (C),which records at least one operating parameter of the opticalarrangement (1) and which adjusts the tilting situation to be dependentof the value of the operating parameter, and wherein the confocal screenis designed as a split diaphragm (4) and the detector unit is designedas detector line (2) and wherein the tilting situation is two-axleadjusted such that the image of the slit diaphragm (4) is centeredtwo-axle on the detector line (2).
 30. Microscope according claim 23,wherein the plate (9) has at least one of a tilting and swivelingmovement around at least two axes, wherein the plate (9) is providedbetween the specimen range (10) and the confocal screen (4) and alsobetween the screen (4) and the detector unit (2).
 31. Microscopeaccording to claim 23, comprising at least two plane-paralleltransparent plates (9), each of the plates (9) being tiltable around oneaxis wherein one of the plates (9) is provided between the specimenrange (10) and the confocal screen (4) and another of the plates (9) isprovided between the screen (4) and the detector unit (2). 32.Microscope according to claim 23 wherein the microscope has anexcitation light path for the illumination of the selected specimenrange (10), and wherein the plane-parallel plate (9) is included in theexcitation light path.